Regulation of protein synthesis at the level of mRNA translation is important for: (1) viral shut-off of host protein synthesis, (2) rapid adjustments to changing metabolic conditions, and (3) coordination of heme-globin-biosynthesis during red blood cell maturation. We have therefore studied the role of eIF-2 phosphorylation and mixed disulfide formation during protein synthesis initiation, and its regulation by the adenylate energy change, the pyridine nucleotide redox state and hemin, in reticulocyte lysate and intact cells. Translational regulation is shown to involve changes in the phosphorylation state and the oxidaton/reduction state of the initiation factor eIF-2. In addition, a new polypeptide complex was isolated which participates in the catlytic utilization of eIF-2. The phosphorylation state of eIF-2 has been measured by chemical methods and the results correlated with the extent and kinetics of onset of translational inhibition. It now appears that phosphorylation of eIF-2 does not directly inhibit its activity but may interfere with the interaction of eIF-2 and the new polypeptide complex. Phosphorylation may promote additional modifications of eIF-2, however, which result in conversion from catalytic to stoichiometric utilization of eIF-2.